Combining enrichment with multiplex real-time PCR leads to faster detection and identification of Campylobacter spp. in food compared to ISO 10272-1:2017.

Conventional protocols for the detection of Campylobacter from foods are laborious and time-consuming. This research describes an alternative procedure (EMRT-PCR) for the detection of Campylobacter from food by combining ISO 10272-1:2017 enrichment in Bolton broth (BB) with a multiplex real-time (MRT-) PCR assay. Species differentiation was done by targeting C. jejuni (mapA), C. coli (ceuE), and both species (cje). The detection limit of the MRT-PCR assay was 4.5 and 5.5 log10 cfu/ml in BB and BB containing chicken skin, respectively. A Monte Carlo simulation was conducted to predict the probability that campylobacters reach the MRT-PCR detection threshold throughout enrichment in BB, and results suggested that cold-stressed campylobacters could reach the detection limit after 40 h of enrichment (p = 0.99). As a proof of principle, 23 naturally contaminated meat products were enriched according to ISO 10272-1:2017 procedure A, and the EMRT-PCR in parallel. After 24 h, 12 and 11 samples already tested positive for Campylobacter with the ISO method and EMRT-PCR, respectively. After 40 h, the 24-h-negative sample was also positive with EMRT-PCR. The EMRT-PCR takes about 2 days to produce reliable results, while results using ISO 10272-1:2017 can take up to 8 days, which demonstrate the potential of the EMRT-PCR method.

Role of substrate availability in the growth of Campylobacter co-cultured with extended spectrum beta-lactamase-producing Escherichia coli in Bolton broth.

It is well-established that Extended-spectrum beta-lactamase-producing (ESBL-) Escherichia coli challenge reliable detection of campylobacters during enrichment in Bolton broth (BB) following ISO 10272-1:2017. The overgrowth of Campylobacter by ESBL-E. coli in the enrichment medium BB can lead to false-negative detection outcomes, but the cause for the growth suppression is yet unknown. A plausible reason could be the competition-induced lack of certain growth substrates. Therefore, this study aimed to investigate whether campylobacters and ESBL-E. coli compete for the same medium components and whether this is the cause for the observed growth repression. The availability of possible growth substrates in BB was determined and changes in their extracellular concentration were measured over time during mono-culture enrichment of C. jejuni, C. coli or ESBL-E. coli as well as in co-culture enrichments of campylobacters and ESBL-E. coli. Comparative analysis showed lactate and fumarate utilization by C. jejuni and C. coli exclusively, whereas ESBL-E. coli rapidly consumed asparagine, glutamine/arginine, lysine, threonine, tryptophan, pyruvate, glycerol, cellobiose, and glucose. Both campylobacters and ESBL-E. coli utilized aspartate, serine, formate, a-ketoglutarate and malate. Trends in compound utilization were similar for C. jejuni and C. coli and trends in compound utilization were rather comparable during enrichment of reference and freeze-stressed campylobacters. Since final cell densities of C. jejuni and C. coli in co-cultures were not enhanced by the addition of surplus l-serine and final cell densities were similar in fresh and spent medium, growth suppression seems not to be caused by a lack of substrates or production of inhibitory compounds. We hypothesized that oxygen availability was limiting growth in co-cultures. Higher oxygen availability increased the competitive fitness of C. jejuni 81-176 in co-culture with ESBL-E. coli in duplicate experiments, as cell concentrations in stationary phase were similar to those without competition. This could indicate the critical role of oxygen availability during the growth of Campylobacter and offers potential for further improvement of Campylobacter spp. enrichment efficacy.

Temperature and presence of ethanol affect accumulation of intracellular trehalose in Lactobacillus plantarum WCFS1 upon pulsed electric field treatment.

Pulsed electric field (PEF) treatment can be used to increase intracellular small molecule concentrations in bacteria, which can lead to enhanced robustness of these cells during further processing. In this study we investigated the effects of the PEF treatment temperature and the presence of 8% (v/v) ethanol in the PEF medium on cell survival, membrane fluidity and intracellular trehalose concentrations of Lactobacillus plantarum WCFS1. A moderate PEF treatment temperature of 21 °C resulted in a high cell survival combined with higher intracellular trehalose concentrations compared to a treatment at 10 and 35 °C. Interestingly, highest intracellular trehalose concentrations were observed upon supplementing the PEF medium with 8% ethanol, which resulted in more than a doubling in intracellular trehalose concentrations, while culture survival was retained. Overall, this study shows that treatment temperature and PEF medium optimization are important directions for improving molecule uptake upon PEF processing.

Variability in lag-duration of Campylobacter spp. during enrichment after cold and oxidative stress and its impact on growth kinetics and reliable detection.

Campylobacter jejuni and Campylobacter coli continue to be the leading cause of zoonotic gastroenteritis in the European Union, making reliable detection in food important. Low storage temperatures and atmospheric oxygen concentrations during food production can cause sub-lethal damage or transient non-culturability which is why ISO 10272-1:2017 includes an enrichment step to repair cell damage and increase cell concentrations, thereby supporting detection of campylobacters from foods. The aim of this study was to assess the variability in lag-duration of C. jejuni and C. coli during enrichment after different food-relevant stress treatments and evaluate its impact on growth kinetics and reliability of detection outcomes. Therefore, 13 C. jejuni and 10 C. coli strains were subjected to cold stress during refrigerated and frozen storage. Refrigerated storage did not significantly reduce culturability, but frozen storage reduced cell concentrations by 1.6 ± 0.1 log10cfu/ml for both species. Subsequently, cells were enriched following ISO 10272-1:2017-A and cell concentrations were determined over time and lag-duration and growth rate were determined by fitting the Baranyi-model. Without prior stress treatment, mean lag-duration for C. jejuni and C. coli was 2.5 ± 0.2 h and 2.2 ± 0.3 h, respectively. Refrigerated storage increased lag-duration for C. jejuni to 4.6 ± 0.4 h and for C. coli to 5.0 ± 0.4 h and frozen storage increased lag-duration to 5.0 ± 0.3 h and 6.1 ± 0.4 h for C. jejuni and C. coli, respectively. Comparison of strain- and biological variability showed that differences in recovery after cold stress can be attributed mainly to strain variability since strain variability after refrigeration and freeze stress increased respectively 3-fold and 4-fold while biological variability remained constant. A subset of strains was also subjected to oxidative stress that reduced cell concentrations by 0.7 ± 0.2 log10 cfu/ml and comparison of recovery patterns after oxidative and freeze stress indicated that recovery behaviour was also dependent on the stress applied. A scenario analysis was conducted to evaluate the impact of heterogeneity in outgrowth kinetics of single cells on the reliability of detection outcomes following ISO protocol 10272-1:2017. This revealed that a 'worst-case'-scenario for successful detection by a combination of the longest lag-duration of 7.6 h and lowest growth rate of 0.47 h-1 still resulted in positive detection outcomes since the detection limit was reached within 32.5 h. This suggests that other factors such as competitive microbiota can act as a causative factor in false-negative outcomes of tested food samples.

Reversibility of membrane permeabilization upon pulsed electric field treatment in Lactobacillus plantarum WCFS1.

Pulsed electric field (PEF) treatment, or electroporation, can be used to load molecules into cells. The permeabilizing effect of the PEF treatment on the cellular membrane can be either reversible or irreversible depending on the severity of the PEF treatment conditions. The influence of PEF on the reversibility of membrane permeabilization in Lactobacillus plantarum WCFS1 by two different fluorescent staining methods was investigated in this study. Whereas staining with propidium iodide (PI) before and after PEF treatment indicated small reversible permeabilized fractions of maximum 14%, the use of a double staining method with PI and SYTOX Green suggested larger reversible permeabilized fractions up to 40% of the population. This difference shows that the choice for a fluorescent staining method affects the conclusions drawn regarding reversibility of membrane permeabilization. Additionally, the effect of PEF treatment conditions on membrane integrity was compared, indicating a relation between critical electric field strength, cell size and membrane permeabilization. Overall this study showed the possibilities and limitations of fluorescent membrane integrity staining methods for PEF studies.

The effect of different matrices on the growth kinetics and heat resistance of Listeria monocytogenes and Lactobacillus plantarum.

Microbial growth and inactivation kinetics in food can be predicted when the effects of food properties and environmental conditions on microbial responses are available. However the effects of these intrinsic and extrinsic variables on microbial kinetics are often obtained using laboratory media, and deviations between predictions and true behaviour might occur if the specific effect of a food product is not known or considered in the prediction. Therefore, knowing the food specific effect on microbial kinetics might not only result in a more realistic growth and inactivation prediction, but also extend the knowledge on factors influencing growth and heat resistance. In this study, growth predictions of Listeria monocytogenes and Lactobacillus plantarum were validated in laboratory media and in milk and ham as model food products. A good agreement between the predicted and observed growth kinetics in laboratory media highlighted the possibility to predict µmax based on cardinal growth parameters obtained from OD-based measurement in laboratory media. Only in two conditions (BHI pH5.5 at 7°C; and BHI pH5.5, undissociated lactic acid concentration of 1mM at 7°C) a possible interaction between growth limiting factors was observed, yet existing interaction models were not better in predicting growth. Growth validation in the two model foods showed that the food specific effects were strain dependent, which might further complicate accurate prediction. For both species the effect of strain variability on thermal inactivation was similar to the food specific effects, and the latter was mainly determined by the effect of ham as heating medium. The combination of both effects explained (almost) all variability found in literature, however, with some bias.

Quantifying Variability in Growth and Thermal Inactivation Kinetics of Lactobacillus plantarum.

UNLABELLED: The presence and growth of spoilage organisms in food might affect the shelf life. In this study, the effects of experimental, reproduction, and strain variabilities were quantified with respect to growth and thermal inactivation using 20 Lactobacillus plantarum strains. Also, the effect of growth history on thermal resistance was quantified. The strain variability in µmax was similar (P > 0.05) to reproduction variability as a function of pH, aw, and temperature, while being around half of the reproduction variability (P < 0.05) as a function of undissociated lactic acid concentration [HLa]. The cardinal growth parameters were estimated for the L. plantarum strains, and the pHmin was between 3.2 and 3.5, the aw,min was between 0.936 and 0.953, the [HLamax], at pH 4.5, was between 29 and 38 mM, and the Tmin was between 3.4 and 8.3°C. The average D values ranged from 0.80 min to 19 min at 55°C, 0.22 to 3.9 min at 58°C, 3.1 to 45 s at 60°C, and 1.8 to 19 s at 63°C. In contrast to growth, the strain variability in thermal resistance was on average six times higher than the reproduction variability and more than ten times higher than the experimental variability. The strain variability was also 1.8 times higher (P < 0.05) than the effect of growth history. The combined effects of strain variability and growth history on D value explained all of the variability as found in the literature, although with bias. Based on an illustrative milk-processing chain, strain variability caused ∼2-log10 differences in growth between the most and least robust strains and >10-log10 differences after thermal treatment. IMPORTANCE: Accurate control and realistic prediction of shelf life is complicated by the natural diversity among microbial strains, and limited information on microbiological variability is available for spoilage microorganisms. Therefore, the objectives of the present study were to quantify strain variability, reproduction (biological) variability, and experimental variability with respect to the growth and thermal inactivation kinetics of Lactobacillus plantarum and to quantify the variability in thermal resistance attributed to growth history. The quantitative knowledge obtained on experimental, reproduction, and strain variabilities can be used to improve experimental designs and to adequately select strains for challenge growth and inactivation tests. Moreover, the integration of strain variability in prediction of microbial growth and inactivation kinetics will result in more realistic predictions of L. plantarum dynamics along the food production chain.

Impact of Pathogen Population Heterogeneity and Stress-Resistant Variants on Food Safety.

This review elucidates the state-of-the-art knowledge about pathogen population heterogeneity and describes the genotypic and phenotypic analyses of persister subpopulations and stress-resistant variants. The molecular mechanisms underlying the generation of persister phenotypes and genetic variants are identified. Zooming in on Listeria monocytogenes, a comparative whole-genome sequence analysis of wild types and variants that enabled the identification of mutations in variants obtained after a single exposure to lethal food-relevant stresses is described. Genotypic and phenotypic features are compared to those for persistent strains isolated from food processing environments. Inactivation kinetics, models used for fitting, and the concept of kinetic modeling-based schemes for detection of variants are presented. Furthermore, robustness and fitness parameters of L. monocytogenes wild type and variants are used to model their performance in food chains. Finally, the impact of stress-resistant variants and persistence in food processing environments on food safety is discussed.

Quantifying strain variability in modeling growth of Listeria monocytogenes.

Prediction of microbial growth kinetics can differ from the actual behavior of the target microorganisms. In the present study, the impact of strain variability on maximum specific growth rate (µmax) (h(-1)) was quantified using twenty Listeria monocytogenes strains. The µmax was determined as function of four different variables, namely pH, water activity (aw)/NaCl concentration [NaCl], undissociated lactic acid concentration ([HA]), and temperature (T). The strain variability was compared to biological and experimental variabilities to determine their importance. The experiment was done in duplicate at the same time to quantify experimental variability and reproduced at least twice on different experimental days to quantify biological (reproduction) variability. For all variables, experimental variability was clearly lower than biological variability and strain variability; and remarkably, biological variability was similar to strain variability. Strain variability in cardinal growth parameters, namely pHmin, [NaCl]max, [HA]max, and Tmin was further investigated by fitting secondary growth models to the µmax data, including a modified secondary pH model. The fitting results showed that L. monocytogenes had an average pHmin of 4.5 (5-95% prediction interval (PI) 4.4-4.7), [NaCl]max of 2.0mM (PI 1.8-2.1), [HA]max of 5.1mM (PI 4.2-5.9), and Tmin of -2.2°C (PI (-3.3)-(-1.1)). The strain variability in cardinal growth parameters was benchmarked to available literature data, showing that the effect of strain variability explained around 1/3 or less of the variability found in literature. The cardinal growth parameters and their prediction intervals were used as input to illustrate the effect of strain variability on the growth of L. monocytogenes in food products with various characteristics, resulting in 2-4 logCFU/ml(g) difference in growth prediction between the most and least robust strains, depending on the type of food product. This underlined the importance to obtain quantitative knowledge on variability factors to realistically predict the microbial growth kinetics.

Statistical aspects of food safety sampling.

In food safety management, sampling is an important tool for verifying control. Sampling by nature is a stochastic process. However, uncertainty regarding results is made even greater by the uneven distribution of microorganisms in a batch of food. This article reviews statistical aspects of sampling and describes the impact of distributions on the sampling results. Five different batch contamination scenarios are illustrated: a homogeneous batch, a heterogeneous batch with high- or low-level contamination, and a batch with localized high- or low-level contamination. These batch contamination scenarios showed that sampling results have to be interpreted carefully, especially when heterogeneous and localized contamination in food products is expected.

Quantifying variability on thermal resistance of Listeria monocytogenes.

Knowledge of the impact of strain variability and growth history on thermal resistance is needed to provide a realistic prediction and an adequate design of thermal treatments. In the present study, apart from quantifying strain variability on thermal resistance of Listeria monocytogenes, also biological variability and experimental variability were determined to prioritize their importance. Experimental variability was defined as the repeatability of parallel experimental replicates and biological variability was defined as the reproducibility of biologically independent reproductions. Furthermore, the effect of growth history was quantified. The thermal inactivation curves of 20 L. monocytogenes strains were fitted using the modified Weibull model, resulting in total 360 D-value estimates. The D-value ranged from 9 to 30 min at 55 °C; from 0.6 to 4 min at 60 °C; and from 0.08 to 0.6 min at 65 °C. The estimated z-values of all strains ranged from 4.4 to 5.7 °C. The strain variability was ten times higher than the experimental variability and four times higher than the biological variability. Furthermore, the effect of growth history on thermal resistance variability was not significantly different from that of strain variability and was mainly determined by the growth phase.

Quantification of the impact of single and multiple mild stresses on outgrowth heterogeneity of Bacillus cereus spores.

Outgrowth heterogeneity of bacterial spore populations complicates both prediction and efficient control of spore outgrowth. In this study, the impact of mild preservation stresses on outgrowth of Bacillus cereus ATCC 14579 spores was quantified during the first stages of outgrowth. Heterogeneity in outgrowth of heat-treated (90°C for 10 min) and non-heat-treated germinated single spores to the maximum micro-colony stage of 256 cells was assessed by direct imaging on Anopore strips, placed on BHI plates at pH7 and pH5.5, without and with added NaCl or sorbic acid (HSA). At pH7 non-heated and heat-treated germinated spores required 6h to reach the maximum microcolony stage with limited heterogeneity, and these parameters were only slightly affected with both types of spores when incubated at pH7 with added NaCl. Notably, the most pronounced effects were observed during outgrowth of spores at pH5.5 without and with added NaCl or HSA. Non-heat-treated germinated spores showed again efficient outgrowth with limited heterogeneity reaching the maximum microcolony stage after 6h at pH5.5, which increased to 12h and 16 h with added NaCl and HSA, respectively. In contrast, heat-treated spores displayed a strong delay between initial germination and swelling and further outgrowth at pH5.5, resulting in large heterogeneity and low numbers of fastest growers reaching the maximum microcolony stage after 10, 12 and 24h, without and with added NaCl or HSA, respectively. This work shows that Anopore technology provides quantitative information on the impact of combined preservation stresses on outgrowth of single spores, showing that outgrowth of germinated heat-treated spores is significantly affected at pH5.5 with a large fraction of spores arrested in the early outgrowth stage, and with outgrowing cells showing large heterogeneity with only a small fraction committed to relatively fast outgrowth.

Prediction of Bacillus weihenstephanensis acid resistance: the use of gene expression patterns to select potential biomarkers.

Exposure to mild stress conditions can activate stress adaptation mechanisms and provide cross-resistance towards otherwise lethal stresses. In this study, an approach was followed to select molecular biomarkers (quantitative gene expressions) to predict induced acid resistance after exposure to various mild stresses, i.e. exposure to sublethal concentrations of salt, acid and hydrogen peroxide during 5 min to 60 min. Gene expression patterns of unstressed and mildly stressed cells of Bacillus weihenstephanensis were correlated to their acid resistance (3D value) which was estimated after exposure to lethal acid conditions. Among the twenty-nine candidate biomarkers, 12 genes showed expression patterns that were correlated either linearly or non-linearly to acid resistance, while for the 17 other genes the correlation remains to be determined. The selected genes represented two types of biomarkers, (i) four direct biomarker genes (lexA, spxA, narL, bkdR) for which expression patterns upon mild stress treatment were linearly correlated to induced acid resistance; and (ii) nine long-acting biomarker genes (spxA, BcerKBAB4_0325, katA, trxB, codY, lacI, BcerKBAB4_1716, BcerKBAB4_2108, relA) which were transiently up-regulated during mild stress exposure and correlated to increased acid resistance over time. Our results highlight that mild stress induced transcripts can be linearly or non-linearly correlated to induced acid resistance and both approaches can be used to find relevant biomarkers. This quantitative and systematic approach opens avenues to select cellular biomarkers that could be incremented in mathematical models to predict microbial behaviour.